1. Field of the Invention
The present invention is directed to a stabilized phenol or phenol-chloroform solution useful, for example, in the purification of nucleic acids when soluble proteins are to be removed from the nucleic acid solution. The solution remains chemically stable under inert gas atmosphere during long-term storage. The present invention further relates to a phenol compatible package which preserves the chemical stability of the solution and safely delivers the phenol.
2. Description of the Prior Art
The isolation and purification of nucleic acids is basic to all procedures in molecular cloning. For example, when DNA is to be used for genetic engineering, that is, when it is to be cut with restriction enzymes, ligated, kinased, and so on, the DNA needs to be scrupulously clean.
One of the first reports of nucleic acid isolation described the preparation of ribonucleic acid (RNA) from a plant virus (the Tobacco Mosaic Virus) using mild procedures. Viral RNA was successfully separated from nucleoproteins after the proteins had been denatured by heating.
However, early attempts at isolating RNA from mammalian tissues were not entirely satisfactory. Unlike nucleoproteins from tobacco mosaic virus, spontaneous dissociation after heat denaturation did not occur with the mammalian RNA-protein complex. This suggested that the RNA-protein complex obtained from mammals is held by a stronger bond than that from tobacco mosaic virus.
To separate the protein from the RNA, it was essential to denature the proteins by heating the nucleoprotein preparation to 90.degree.-100.degree. C. Since the cell nucleus is the site of genetic material and therefore of DNA, these early methods also involved separation of cell nuclei from the cytoplasm in order to obtain DNA-free cell lysates. This practice, along with other drastic procedures generally used in isolating RNA from mammalian tissues probably caused degradation of RNA. Consequently, early RNA yields were of low molecular weight and were not highly polymerized.
In time, through a lengthy precipitation and ultra centrifugation method it was possible to split off the protein moiety from a glycoprotein by treatment with phenol and water, yielding polysaccharides and RNA's in the aqueous phase. By modifying this method it was also possible to extract mammalian RNA's from nucleoproteins at 68.degree. C. Still further investigations showed that better results could be obtained by performing the extraction at room temperature. (K. S. Kirby, Biochemistry Journal, Vol 64, p. 405 (1956)).
In 1956, Kirby devised a method of preparing RNA from mammalian tissues by extraction with phenol and water at room temperature. Kirby found that phenol was essential to the process and concluded that phenol's properties as a protein solvent were important in the separation. The Kirby method had several advantages. Firstly, the deoxyribonucleic acids (DNA's), remained completely insoluble and therefore separable from RNA, thus making it no longer necessary to separate the nuclei before preparing RNA. Secondly, the phenol treatment inactivated ribonucleases in the tissue, and possibly other nucleases as well, thus yielding highly polymerized RNA's.
In 1957, Kirby reported that by modifying his RNA separation technique, he was able to successfully liberate DNA as well. (K. S. Kirby, Biochemistry Journal, Vol 66, p. 495, 501 (1957)). By replacing the water in the original phenol-water mixture with solutions of certain anions, DNA as well as RNA was brought into the aqueous layer. It was essential to homogenize the tissue in a salt solution first and then treat with phenol, since no DNA was released by treating tissues with water and phenol and subsequently adding the effective salts. Phenol, acting as a protein solvent, was as essential to the reactions as the salt in solution.
When mammalian tissues are treated with a salt solution and phenol as described above, a DNA-protein complex is liberated. DNA can then be freed completely from protein by using p-aminosalicylate and phenol resulting in a high DNA yield. Also, DNA can be freed completely from RNA by treatment with ribonuclease and precipitation with 2-ethoxyethanol.
The described Kirby method is the basis for current laboratory techniques employed in the separation of RNA and DNA from mammalian tissues. At the present time, when nucleic acids are to be purified from a complex mixture of molecules such as cell lysates, the usual method is to remove most of the protein by digesting with proteolytic enzymes such as pronase or proteinase-K, which are active against a broad spectrum of native proteins. The nucleic acid is subsequently isolated. A key step in the final isolation of nucleic acid is the removal of proteins, a process often carried out by extracting aqueous solutions of nucleic acid with organic solvents such as phenol and/or chloroform.
The removal of proteins from nucleoproteins, the key step of nucleic acid purification, may be carried out by using phenol, but there are two major disadvantages. First, it is crucial to remove from the phenol all contaminants that would bring about cross-linking of RNA and DNA. That is, phenol must be chemically stable and non-reactive with other elements in its environment. Current laboratory manuals describe a lengthy process by which this may be accomplished. (See, for example, T. Maniatis, E. F. Fritsch, J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Publications, (1982)). However, stabilized phenol solutions must either be prepared as needed or else be stored at 4.degree. C. under equilibration buffer. Under the latter conditions, the solution remains stable only for periods of up to one month.
Second, phenol is highly corrosive and can cause severe burns. It is recommended that safety glasses and gloves be worn while handling phenol. Maniatis, supra, at 438.
In summary, an aqueous phenol solution is regularly used in the purification of nucleic acids, but for this purpose, it must be chemically stable and free of contaminants. Under present laboratory methods, phenol solution becomes unstable shortly after it is prepared. During preparation, extreme caution must be exercised because phenol is highly corrosive.